Chapter 5: Effects of stem training on physiology, growth, and yield responses of
5.3 Materials and methods
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experimental units (3 x 3 x 4). Six weeks old seedlings were used and transplanted into 8 L bags filled with fine pine sawdust as a growing medium. The single stem (SS) training method was achieved by planting one seedling in a pot and removing all sucker stem as the plant grow, to allow plants to grow as a single leader. On the other hand, two plants per pot (TPP) method was achieved by planting two seedlings in one pot and remove all sucker stem to allow each seedling to grow as a single stem. Plants assigned to double stem (DS) were achieved by planting one seedling in a pot and allow the sucker at the bottom to grow as the second main stem resulting to double leader stems growing. Water-soluble inorganic fertilizer mix (commercial fertilizer) in the form of Solucal® (calcium nitrate), Multi-K (potassium nitrate) and Hygroponic® (ammonium nitrate with all essential micronutrients and macronutrients) was dissolved in one tank filled with 5000 L of water. Fertilizers were mixed according to the recommended rate for tunnel production by the manufacturer (Hygrotech SA, Pietermaritzburg, South Africa). A 2.7 kg Solucal, 500 g multi-K®
and 3 kg Hygroponic® was mixed with water in a 5000 L tank from transplant to third flower.
After the third flower truss to the end, fertilizers were increased by mixing 5000 L of water with 3.5 kg Solucal®, 1 kg Multi-K® and 5 kg Hygroponic®.
5.3.4 Data collection
5.3.4.1 Measurement of leaf gaseous exchange parameters
Leaf gas exchange was measured on Week 3, 10, 12, 14, 18 after planting, using Portable Photosynthesis System LI-6400 XT (Licor Bioscience, Inc. Lincoln, Nebraska, USA) fitted with infrared gas analyzer connected to a leaf chamber fluorimeter (LCF) (6400-40B, 2 cm2 leaf area, Licor Bioscience, Inc. Lincoln, Nebraska, USA). The artificial saturated photosynthetic active
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radiation (PAR) and external CO2 were fixed at 1000 µmol m-2 s-1 and 400 µmol mol-1, respectively. The measurements were taken at two weeks’ intervals on sunny days between 11h00 and 13h00. Sampling was taken in the apex of one leaf of each plant representing a replicate. Leaf gaseous exchange parameters such as photosynthetic rate (A), stomatal conductance (gs), transpiration rate (T), intercellular CO2 concentration (Ci) and the ratio of intercellular and atmospheric CO 2 (Ci/Ca) concentration were measured. The stomatal limitation was calculated as 1-Ci/Ca (Dong et al., 2016). Water use efficiency (WUE) was calculated as the ratio of A and T (Mashilo et al., 2017).
5.3.4.2 Plant growth parameters and yield
Plant height was measured on a 2-week interval, using a measuring tape. Measurements were taken from the base up to the apical point of the plant. Samples were taken in was made in each replicate of all the treatments. Stem diameter was measured using a caliper. Measurements were taken at the base of the stem in each plant representing a replicate. The yield of tomato was determined by the number of fruit harvested and mass measured. Fruit sampling for yield and quality measurement was taken on three sampling dates denoted as Harvest 1, 2 and 3. The number of fruit was determined by counting. On the other hand, fruit mass was determined by weighing individually fruit using a calibrated benchtop balanced weighing scale (WTB200, RADWAG.
Poland). The sum of all fruit harvested and mass was used to estimate total yield.
5.3.4.3 Total soluble solids (TSS) and Titratable acid (TA)
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TSS was measured using a benchtop digital refractometer (RFM 340 +, Bellingham + Stanley Ltd, UK) based on a method described by Ncama et al. (2017). Fruit juice was obtained by crushing a fruit using a Warring blender, followed by squeezing fruit juice into 50 mL beaker using a nylon filter. The refractometer was calibrated by cleaning a prism with distilled water, followed by wiping with a clean paper towel and measuring a zero sample. After calibration, tomato juice of each fruit representing a replicate was measured to determine a TSS.
TA was measured using Mettler Toledo compact titrator G10S. Briefly, samples were prepared by pipetting 8 mL of juice into a 100 mL beaker. Using another clean pipette tip, 42 mL distilled water was added to the juice in the beaker and titrated with 0.1M NaOH to a pH value of 8.1 using a Mettler Toledo. The acid was calculated as the percentage of citric acid using a factor 0.0064, Eq. 1.
Percentage acid=𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡(𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚)𝑋𝑋𝐴𝐴𝑋𝑋𝑋𝑋𝑋𝑋𝑋𝑋𝑚𝑚𝑋𝑋𝑡𝑡𝑋𝑋𝑡𝑡𝑋𝑋100
8(𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑡𝑡𝑋𝑋𝑡𝑡) 1
TA was measured in each fruit per plant, per replicate, per treatment and taking the average mean of replicates.
BrimA (Brix minus Acid) an index that measures the balance between sweetness and acidity. It was calculated using Eq. 2 suggested by Jordan et al. (2001).
BrimA = TSS − 𝑘𝑘 (TA) 2
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Where k is a constant that reflects the tongue's less sensitivity to TSS compared to TA. The k constant allows the TSS amounts higher than TA to make the same numerical change to BrimA.
The equation of BrimA (Eq. 2) was adjusted as recommended by Obenland et al. (2009) who had replaced the constant (k) value 5 as suggested by Jordan et al. (2001) with 3 and 4 to eliminate the generation of negative BrimA values for oranges.
5.3.4.4 Fruit colour
Colour of tomato fruit was measured using Konica Minolta Chroma meter CR-300, INC, Japan (López Camelo and Gómez, 2004). Measurements were taken on the equatorial region of the fruit.
Fruit samples were scanned on three parts and reading on the chromameter. Colour co-ordinates readings recorded, lightness (L*), green to red (a*) and blue to yellow (b*) and the results combined as the Tomato Colour Index (Hobson et al., 1983) using Eq. 3
Colour index = 2000𝑎𝑎 ÷ 𝐿𝐿𝐿𝐿
5.3.5 Statistical analysis
The collected data of measured variables were subjected to the analysis of variance (ANOVA) using statistical software GenStat (GenStat1, 18.1 edition, VSN International, UK). Mean was separated using Fischer’s least significant difference (LSD) at 5% level of significance. The values of Standard error were calculated where a significant standard deviation was found at (p < 0.05)
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between individual values.Pearson's correlation analysis was performed to describe the pattern of relationship between plant growth and leaf gas exchange parameters using Excel window’s 10.
Significance tests of the correlation coefficients were determined using excel window’s 10.